The p53 gene is one of the most studied and well-known genes. p53 plays a key role in cellular stress response mechanisms by converting a variety of different stimuli, for example, DNA damage, deregulation of transcription or replication, and oncogene transformation, into cell growth arrest or apoptosis (Kastan et al., Cancer Res 1991; 51:6304-6311; Vogelstein et al., Nature 2000; 408:307-310; Vousden et al., Nat Rev Cancer 2002; 2:594-604; Giaccia et al., Genes & Development 1998; 12:2973-2983; T. M. Gottlieb et al., Biochem. Biophys. Acta, 1287, p. 77 (1996)).
The p53 protein is active as a homo-tetramer and exerts its tumor suppressor function mainly as a transcription factor that affects G1 and G2 cell cycle arrest and/or apoptosis (see, e.g., Donehower and Bradley, Biochim Biophys Acta., 1993, 1155(2):181-205; Haffner and Oren, Curr. Opin. Genet. Dev., 1995, 5(1):84-90; Gottlieb and Oren, Biochim. Biophys. Acta., 1996, 1287(2-3):77-102; Ko and Prives, Genes Dev., 1996, 10(9):1054-72; Hansen and Oren, Curr. Opin. Genet. Dev., 1997, 7(1):46-51; Levine, Cell, 1997, 88(3):323-31). The p53-mediated G1 arrest is its best characterized activity and involves transcriptional activation of the downstream gene p21 WAF1/CIP1/SDI1 (Haffner and Oren, Curr. Opin. Genet. Dev., 1995, 5(1):84-90; Gottlieb and Oren, Biochim. Biophys. Acta., 1996, 1287(2-3):77-102; Ko and Prives, Genes Dev., 1996, 10(9):1054-72; Hansen and Oren, Curr. Opin. Genet. Dev., 1997, 7(1):46-51; Levine, Cell, 1997, 88(3):323-31). Other downstream effector genes for p53-mediated G1 arrest may exist, since p21−/− mouse embryonic fibroblasts do not show complete abrogation of G1 arrest after DNA damage (Brugarolas et al., Nature, 1995, 377(6549):552-7; Deng et al., Cell, 1995, 82(4):675-84). The G2/M effects of p53 involve, at least in part, induction of 14-3-3σ (Hermeking et al., Mol. Cell, 1997, 1(1):3-11).
The mechanisms for apoptosis induction and their relative importance remain less clear at present. In certain settings p53 clearly induces pro-apoptotic genes. These include BAX and Fas/APO1 (Miyashita and Reed, Cell, 1995, 80(2):293-9; Owen-Schaub et al., Mol. Cell. Biol., 1995, 15(6):3032-40) neither of which, however, is an absolute requirement for p53-induced apoptosis (Fuchs et al., Cancer Res., 1997, 57(13):2550-4). Recently, many more genes have been identified that are induced directly or indirectly during p53-mediated apoptosis (Polyak et al., Nature, 1997, 389(6648):300-5), but the essential genes for p53-induced apoptosis still have to be determined. Transcriptional repression of anti-apoptotic genes, such as bcl-2, may play a role (Haldar et al., Cancer Res., 1994, 54(8):2095-7; Miyashita et al., Oncogene, 1994, 9(6):1799-805) and other non-transcriptional mechanisms may be important as well (Caelles et al., Nature, 1994, 370(6486):220-3; Haupt et al., Nature 1997; 387:296-299).
Several upstream signals activate p53. These include DNA damage, hypoxia and critically low ribonucleoside triphosphate pools (Kastan et al., Cancer Res. 1991; 51:6304-6311; Graeber et al., Nature, 1996, 379(6560):88-91; Linke et al., Genes Dev., 1996, 10(8):934-47). Once activated, p53 induces either cell cycle arrest or apoptosis, depending on several factors such as the amount of DNA damage, cell type and cellular milieu, e.g., presence or absence of growth factors (Donehower and Bradley, Biochim Biophys Acta., 1993, 1155(2):181-205; Haffner and Oren, Curr. Opin. Genet. Dev., 1995, 5(1):84-90; Gottlieb and Oren, Biochim. Biophys. Acta., 1996, 1287(2-3):77-102; Ko and Prives, Genes Dev., 1996, 10(9):1054-72; Hansen and Oren, Curr. Opin. Genet. Dev., 1997, 7(1):46-51; Levine, Cell, 1997, 88(3):323-31).
Cancer cells show decreased fidelity in replicating their DNA, often resulting in DNA damage, and tumor masses have inadequate neovascularization leading to ribonucleoside triphosphate or oxygen deprivation, all upstream signals that activate p53. In view of p53's capability to induce cell cycle arrest or apoptosis under these conditions it is not surprising that absent or significantly reduced activity of the tumor suppressor protein p53 is a characteristic of more than half of all human cancers (Harris and Hollstein, N. Engl J. Med., 1993, 329(18):1318-27; Greenblatt et al., Cancer Res., 1994, 54(18):4855-78). In the majority of cancers, p53 inactivation is caused by missense mutations in one p53 allele, often with concomitant loss-of-heterozygosity (Michalovitz et al., J. Cell. Biochem., 1991, 45(1):22-9; Vogelstein and Kinzler, Cell, 1992, 70(4):523-6; Donehower and Bradley, Biochim. Biophys. Acta., 1993, 1155(2):181-205; Levine, Cell, 1997, 88(3):323-31). These mutations affect almost exclusively the core DNA-binding domain of p53 that is responsible for making contacts with p53 DNA-binding sites, while mutations in the N-terminal transactivation domain or the C-terminal tetramerization domain are extremely rare (Beroud and Soussi, Nucleic Acids Res., 1998, 26(1):200-4; Cariello et al., Nucleic Acids Res., 1998, 26(1):198-9; Hainaut et al., P., Nucleic Acids Res. 1998; 26:205-213).
Contrary to wild-type p53, p53 cancer mutants have a long half-life and accumulate to high levels in cancer cells (Donehower and Bradley, Biochim Biophys Acta., 1993, 1155(2):181-205; Lowe, Curr. Opin. Oncol., 1995, 7(6):547-53). This may be explained by their inability to activate the mdm-2 gene (Lane and Hall, Trends Biochem. Sci., 1997, 22(10):372-4.), since mdm-2 induces degradation of p53 via the ubiquitin pathway as part of a negative feedback loop (Haupt et al., Nature 1997; 387:296-299; Kubbutat et al., Nature 1997; 387:299-303). The unusually high frequency of p53 missense mutations in human cancers (as opposed to mutations resulting in truncated proteins) is explained by their dominant-negative effect that depends on the intact C-terminal tetramerization domain. The C-terminus allows p53 cancer mutants to form hetero-tetramers with wild-type p53 (Milner and Medcalf, Cell, 1991, 65(5):765-74), thus reducing, or even abrogating, the activity of wild-type p53 protein (Michalovitz et al., J. Cell. Biochem., 1991, 45(1):22-9; Vogelstein and Kinzler, Cell, 1992, 70(4):523-6; Ko and Prives, Genes Dev., 1996, 10(9):1054-72). In addition, there is evidence that at least some of the same missense mutations may confer a gain-of-function (Gottlieb and Oren, Biochim. Biophys. Acta., 1996, 1287(2-3):77-102; Ko and Prives, Genes Dev., 1996, 10(9):1054-72; Levine, Cell, 1997, 88(3):323-31).
p53 has a short half-life, and, accordingly, is continuously synthesized and degraded in the cell. However, when a cell is subjected to stress, p53 is stabilized. Examples of cell stress that induce p53 stabilization are: a) DNA damage, such as damage caused by UV (ultraviolet) radiation, cell mutations, chemotherapy, and radiation therapy; b) hyperthermia; and c) deregulation of microtubules caused by some chemotherapeutic drugs, e.g., treatment using taxol or Vinca alkaloids.
When activated, p53 causes cell growth arrest or a programmed, suicidal cell death, which in turn acts as an important control mechanism for genomic stability. In particular, p53 controls genomic stability by eliminating genetically damaged cells from the cell population, and one of its major functions is to prevent tumor formation.
The p53 gene is commonly mutated in human cancers (Levine et al., Br. J. Cancer 1994; 69:409 and Thompson et al., Br. J. Surg. 1998; 85:1460; Hainaut et al., P., Nucleic Acids Res. 1998; 26:205-213) and inherited mutations in the gene lead to the profound cancer predisposition Li-Fraumeni syndrome (Malkin et al., Science 1990; 250:1233-1238). Loss of the p53 gene in combination with loss of one or more additional tumor suppressor genes is associated with malignant tumor progression. For example, loss of both the p53 gene and the tumor suppressor PTEN are associated with advanced stages of prostate cancer (Di Cristofano et al, Cell 2000; 100:387-390; Vogelstein et al., Nature 2000; 408:307-310). As shown by Chen et al (Nature 2005; 436:725-730), the loss of PTEN alone leads to increased p53 levels and induction of a cellular senescence program for tumor suppression in the PTEN-deficient, neoplastic tissue, while subsequent loss of p53 following PTEN loss removes the senescent signal and leads to aggressive tumor growth. Thus, treatment of early stages of PTEN-deficient prostate neoplasia may benefit from p53 activation in favor of programmed cellular senescence to suppress tumor progression.
The reason that inherited or sporadic mutations in the p53 gene contribute to the development of malignancies is presumably related to its cellular stress response functions. Failure to induce appropriate growth arrest or apoptosis after DNA damage is thought to promote genetic instability or inappropriate survival of damaged cells. Thus, an inability to activate p53 function after DNA damage or other cellular stresses can contribute to the generation of viable, genetically altered cells that can lead to malignancy. A loss or inactivation of p53, therefore, is associated with a high rate of tumor progression and a resistance to cancer therapy. Therefore, conventional theories dictate that suppression of p53 would lead to disease progression and protection of the tumor from a cancer therapy.
Importantly, however, p53 also imparts a high sensitivity to several types of normal tissue subjected to genotoxic stress. Specifically, radiation therapy and chemotherapy exhibit severe side effects, such as severe damage to the lymphoid and hematopoietic system and intestinal epithelia, which limit the effectiveness of these therapies. Other side effects, like hair loss, also are p53 mediated and further detract from cancer therapies. These side effects are caused by p53-mediated apoptosis, which maps tissues suffering from side effects of cancer therapies. Therefore, to eliminate or reduce adverse side effects associated with cancer treatment, it would be beneficial to inhibit p53 activity in normal tissue during treatment of p53-deficient tumors, and thereby protect normal tissue (Komarova et al., Seminars in Cancer Biology 1998; 8(5):389-400).
In summary, p53 has a dual role in cancer therapy. On one hand, p53 acts as a tumor suppressor by mediating apoptosis and growth arrest in response to a variety of stresses and controlling cellular senescence. On the other hand, p53 is responsible for severe damage to normal tissues during cancer therapies. The damage caused by p53 to normal tissue made p53 a potential target for therapeutic suppression. In addition, because more than 50% of human tumors lack functional p53, suppression of p53 would not affect the efficacy of a treatment for such tumors, and would protect normal p53-containing tissues. It has been recognized, however, that therapeutic p53 inhibition should be reversible as long-term p53 inactivation can significantly increase the risk of cancer. For further details on suppression of p53 see, e.g., U.S. Pat. Nos. 6,593,353 and 6,420,136.
The adverse effects of p53 activity on an organism are not limited to cancer or cancer therapies. p53 is activated as a consequence of a variety of stresses associated with injuries (e.g., burns), naturally occurring diseases (e.g., hyperthermia associated with fever, and conditions of local hypoxia associated with a blocked blood supply, stroke, and ischemia) and cell aging (e.g., senescence of fibroblasts). p53 inhibition, therefore, also can be therapeutically effective, for example, in reducing or eliminating p53-dependent neuronal death in the central nervous system (e.g., after brain and spinal cord injury), reducing or eliminating neuronal damage during seizures, suppressing tissue aging, or preservation of tissues and organs prior to transplantation.
p53 regulation has also been shown to affect the pathogenesis of neurodegenerative diseases. For example, as shown by Bae et al. (Neuron 2005; 47:29-41), (i) p53 levels are increased in the brains of mutant huntingtin protein (mHtt) transgenic mice (mHtt-Tg) and Huntington's Disease (HD) patients and (ii) upregulation of p53 transcriptional activity and nuclear p53 levels by mHtt leads to mitochondrial depolarization and cytotoxicity in neuronal cell cultures, revealing a role for p53 regulation in the development of HD. Reduction or elimination of p53 suppresses this neurodegenerative effect. Thus, p53 regulation can be beneficial for amelioration of HD and other neurodegenerative diseases.
Optimal induction of growth arrest or apoptosis after DNA damage requires an increase in the intracellular levels of functional p53 protein (Canman et al., Oncogene 1998; 16:957-966; Canman et al., Genes & Dev. 1995; 9:600-611; Kuerbitz et al, Proc Natl Acad Sci 1992; 89:7491-7495). The increases in p53 protein levels are dependent on the ATM protein kinase after ionizing irradiation (IR) (Kastan et al., Cell 1992; 71:587-597) and on the ATR protein kinase after UV irradiation and many other types of cellular stress (Tibbetts et al., Genes & Development 1999; 13:152-157; Hammond et al., Mol Cell Biol. 2002; 22:1834-1843; Wright et al., Pro Natl Acad Sci U.S.A. 1998; 95:7445-7450). There is a measurable increase in the half-life of p53 protein after DNA damage (Maltzman et al., Molec and Cell Biol 1984; 4(9):1689-1694; Price et al., Oncogene 1993; 8:3055-3062; Maki et al, Mol. Cell. Biol. 1997; 17:355-363) and the increases in cellular p53 protein levels have largely been attributed to this change in half-life. p53 protein is normally a very short-lived cellular protein with rapid proteosomal degradation in unperturbed cells. The HDM2 protein (MDM2 in mice) directly binds to p53 protein (Momand et al., Cell 1992; 69:1237-1245; Oliner et al., Nature 1993; 362:857-860) and functions as an E3 ubiquitin ligase to facilitate the degradation of p53 (Fang et al., S., J Biol Chem 2000; 275:8945-8951; Honda et al., FEBS Letters 1997; 420:25-27; Haupt et al., Nature 1997; 387:296-299; Kubbutat et al., Nature 1997; 387:299-303). Post-translational modifications of HDM2 and p53 after DNA damage appear to inhibit the ability of HDM2 to bind to p53 (Mayo et al., Cancer Research 1997; 57:5013-5016; Khosravi et al., PNAS 1999; 96:14973-14977; Maya et al., Genes & Development 2001; 15:1067-1077; Shieh et al., Cell 1997; 91:325-334; Ashcroft et al., Molecular & Cellular Biology 1999; 19:1751-1758), thus decreasing the proteasomal degradation of p53 protein and increasing cellular levels of the protein. Similarly, induction of the ARF tumor suppressor by oncogenes and other cellular signals leads to increases in p53 protein levels by ARF protein binding to HDM2 and inhibiting HDM2-mediated degradation of p53 (Palmero et al., Nature 1999; 395:127; Kamijo et al., Proc. Natl. Acad. Sci. U.S.A 1998; 95:8292-8297; Sherr et al., Curr. Opin. Genet. Dev. 2000; 10:94-99; Pomerantz et al., Cell 1998; 92:713-723; Stott et al., EMBO J. 1998; 17:5001-5014). Thus, cells with overexpressed HDM2 or inactive ARF are similar to cells containing mutated p53 genes in that normal p53 regulation is lacking.
Several reports have suggested that translational regulation may also contribute to p53 induction after DNA damage. In the initial reports of p53 induction after ionizing irradiation, the protein synthesis inhibitor cycloheximide was shown to block p53 induction and marked increases in labeling of p53 protein with [35S]-methionine were noted early after treatment (Kastan et al., Cancer Res 1991; 51:6304-6311; Kastan et al., Cell 1992; 71:587-597). Subsequently, a translation suppressor element was reported in the 3′UTR of the p53 mRNA (Fu et al., Embo J 1997; 16:4117-4125; Fu et al., Oncogene 1999; 18:6419-6424; Fu et al., EMBO J. 1996; 15:4392-4401) and a stem loop structure was predicted in the 5′UTR of the murine p53 gene (Mosner et al., EMBO J. 1995; 14:4442-4449). Interestingly, p53 was suggested to negatively regulate its own translation by direct binding of p53 protein to this 5′UTR stem loop structure (Mosner et al., EMBO J. 1995; 14:4442-4449). Two other proteins have also been reported to modulate p53 translation: thymidylate synthase suppresses p53 translation by binding to the coding sequence of p53 mRNA (Chu et al., Mol. Cell. Biol. 1999; 19:1582-1594; Ju et al., Proc. Natl. Acad. Sci. U.S.A 1999; 96:3769-3774) and HuR (Hu antigen R) enhances the translation efficiency of p53 after ultraviolet irradiation by binding to an AU-rich sequence at the 3′UTR of p53 mRNA (Mazan-Mamczarz et al., Proc. Natl. Acad. Sci. U.S.A 2003; 100:8354-8359).
Despite suggestions that translational control of p53 might be important, the extent, importance and mechanism of p53 translational regulation after DNA damage has remained unclear.